Process for preparing isomalto-oligosaccharides with elongated chain and low glycemic index

ABSTRACT

The invention relates to a process for preparing isomalto-oligosaccharides with elongated chain length. Isomalto-oligosaccharides are directly converted to isomalto-oligosaccharides with elongated chain length in the presence of glucansucrase. Said products can be applied in food, feed, beverages, cosmetics or pharmaceutical products and are particularly useful as slow or non-digestible oligosaccharides, low calorie providers, prebiotics, mineral absorption promoting agents, non-cariogenic agents and/or low glycemic index regulating syrups.

This Application is the National Phase of International Application No.PCT/EP04/001060 filed Feb. 5, 2004, which designated the U.S. and waspublished under PCT Article 21(2) in English, and this applicationclaims, via the aforesaid International Application, the foreignpriority benefit of and claims the priority from United KingdomApplication No. GB 0302894.1, filed Feb. 8, 2003, the completedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a process for preparingisomalto-oligosaccharides with elongated chain length and low glycemicindex.

BACKGROUND OF THE INVENTION

Isomalto-oligosaccharides such as isomaltotriose and isomaltotetraoseare known as growth-promoting saccharides for bifid bacteria and theywill stimulate, in the large intestine, the growth of bacteria that aresupposed to be beneficial for the health of the individual. The genusBifidobacteria, in particular, is known to have a positive influence onthe general health condition.

It is further described that the bifidus stimulating effect (i.e. theincrease in bifidus population per weight unit ofisomalto-oligosaccharide taken orally) is increased when going from asyrup rich in isomaltose (a disaccharide) towards isomaltotriose- andisomaltotetraose-containing syrups.

Consequently the most potent bifidus stimulatingisomalto-oligosaccharide syrups will comprise of a high content oflonger chain branched oligosaccharides like isomaltotriose,isomaltotetraose, isomaltopentaose, isomaltohexaose and higheranalogues.

U.S. Pat. No. 4,649,058 relates to a process for preparing agluco-oligosaccharide with elongated chains by reacting mono- ordisaccharides as acceptors with sucrose in the presence of α-glucosyltransferase.

So far, the existing processes always start from a pure acceptor withlow molecular weight, said acceptors being at most disaccharides.

There is therefore a need for a process which allows the use of mixtureswherein the acceptor is not limited by its molecular weight. The currentinventor provides such a process.

SUMMARY OF THE INVENTION

The current invention relates to a process for elongating the chainlength of isomalto-oligosaccharides characterised in that said processcomprises an enzymatic transfer reaction between sucrose andisomalto-oligosaccharides present in a carbohydrate syrup. The enzymatictransfer reaction takes place in the presence of glucansucrase, whereinthe glucansucrase is preferably selected from the group consisting ofdextransucrase, altemansucrase, mutansucrase and mixtures thereof.

The invention further relates to a process comprising the followingsteps:

-   -   a) Using one or more isomalto-oligosaccharide producing enzymes        to convert a malto-oligosaccharide syrup into a carbohydrate        syrup containing isomalto-oligosaccharides,    -   b) Adding sucrose to the carbohydrate syrup containing        isomalto-oligosaccharides,    -   c) Adding glucansucrase,    -   d) Converting the isomalto-oligosaccharides in the carbohydrate        syrup into isomalto-oligosaccharides with elongated chain length        for obtaining a syrupy mixture.

In particular, the invention relates to a process wherein theisomalto-oligosaccharide producing enzyme is transglucosidase.

Preferably, the current invention relates to a process wherein, betweenstep a) and b), the carbohydrate syrup containingisomalto-oligosaccharides is purified by chromatography in order toremove glucose therefrom.

The current invention further relates to a process comprisingchromatographic purification of a syrupy mixture containingisomalto-oligosaccharides with elongated chain length.

In a specific embodiment, the process step in the presence of one ormore isomalto-oligosaccharide producing enzymes and/or the process stepin the presence of glucansucrase is (or are) performed on a re-usablecarrier.

Furthermore, the current invention relates to a syrup containingisomalto-oligosaccharides with elongated chain length which isobtainable by the previously described process.

Additionally, the current invention relates to the use of the syrupcontaining isomalto-oligosaccharides with elongated chain length infood, beverages, feed, cosmetics or pharmaceutical products.

It can be used as a slowly or non-digestible oligosaccharides, a lowcalorie provider, a prebiotic, a mineral absorption promoting agent, anon-cariogenic agent and/or a low glycemic index regulating syrup.

DETAILED DESCRIPTION

The current invention relates to a process for elongating chain lengthof isomalto-oligosaccharides characterised in that it comprises anenzymatic transfer reaction between sucrose andisomalto-oligosaccharides present in a carbohydrate syrup. The term“isomalto-oligosaccharide” as used herein refersisomalto-oligosaccharides comprising three or more monosaccharide units.The transfer reaction directly on isomalto-oligosaccharides immediatelyresults in isomalto-oligosaccharides with elongated chain length.

The enzymatic transfer reaction takes place in the presence ofglucansucrase. The glucansucrase is preferably selected from the groupconsisting of dextransucrase, alternansucrase, mutansucrase and mixturesthereof. By applying alternansucrase or mutansucrase, oligosaccharideswith different linkages can be obtained.

The current invention further relates to a process comprising thefollowing steps:

-   -   a) Using one or more isomalto-oligosaccharide producing enzymes        for converting a malto-oligosaccharide syrup into a carbohydrate        syrup containing isomalto-oligosaccharides,    -   b) Adding sucrose to the carbohydrate syrup;    -   c) Adding glucansucrase,    -   d) Producing a syrupy mixture by converting the        isomalto-oligosaccharides in the carbohydrate syrup into        isomalto-oligosaccharides with elongated chain length.

The isomalto-oligosaccharide producing enzyme can be selected fromAspergillus sp. strains (e.g. niger, oryzae, foetidus, carbonarius andarvamori), Aureobasidium sp. strains (e.g. Pullulans), Moniella sp.strains, Brettanomyces sp. strains, Debaryomyces sp. strains,Aspergillus sp. strains, Rhizopus sp. strains, Saccharomyces sp.strains, Leuconostoc sp. and Streptococcus sp. In fact, anyglucosyltransferase or α-glucosidase which allows the conversion ofmalto-oligosaccharides into isomalto-oligosaccharides can be used.

In particular, the present invention relates to a process wherein theisomalto-oligosaccharide producing enzyme is transglucosidase, wherebystep a) is comparable to the enzymatic reaction described in EP 0 875585.

The procedure consists, in a first step, of producingisomalto-oligosaccharides with a transglucosidase which acts onmalto-oligosaccharide syrups (i.e. syrups containing maltose and/ormaltotriose). During the synthesis of isomalto-oligosaccharides frommaltose syrups in the presence of transglucosidase, a considerableamount (15-40% on total carbohydrate weight) of glucose is produced.When the desired composition of isomalto-oligosaccharides is obtained,sucrose and dextransucrase are added to the carbohydrate syrup toproduce longer isomalto-oligosaccharide molecules (starting from theisomalto-oligosaccharides present in the carbohydrate syrup).

Since sucrose is no substrate for the transglucosidase, onlydextransucrase will utilise the sucrose to elongate theisomalto-oligosaccharides.

In order to purify the isomalto-oligosaccharides and remove theconsiderable amounts of glucose formed during the transfer reaction, achromatography can be applied between step a) and b) of current process.

The current invention further relates to a process comprisingchromatographic purification of the syrupy mixture containingisomalto-oligosaccharides with elongated chain length.

In a specific embodiment, the process step in the presence of one ormore isomalto-oligosaccharide producing enzymes and/or the process stepin the presence of glucansucrase is performed on a re-usable carrier. Infact, the industrial viability is further improved by applying the oneor more isomalto-oligosaccharide producing enzymes and/or theglucansucrase in immobilised form. The enzymes may be available inimmobilised form such as cross-linked enzyme crystals. The enzymes canbe immobilised on a carrier, more preferably immobilised on a re-usablecarrier.

For the present purpose re-usable means that the carrier can be freed ofenzyme or enzymatic activity in such a way that the carrier materialstays intact. The carrier may be either continuously or intermittentlyrefreshed. The carrier material can be re-loaded with enzyme andre-used. The cleaning of the carrier can, for example, be performed bywashing with acidic or basic solution. This may be done in batch or inthe column. It may be advantageous to add a salt Another possibility isthe use of protein degrading enzymes. Yet another means would be heatingof the material.

Preferably, the carrier is inert in the sense that it should not affectthe conversion of the substrate into conversion product. Furthermore,the carrier is preferably described as being porous and substantiallynon-compressible. The term ‘porous’ is intended to mean that the solidcarrier comprises a multitude of hollows and pores providing a largesurface area. An example of the use of a porous material is themagnetically stabilised fluidised bed enzyme reactor. The term‘substantially non-compressible’ is intended to mean that the solidcarrier does not deform to any appreciable extent at the pressure whichmight prevail during the conversion process.

Preferably, materials are used which have anion exchange groups. Suchmaterials may be on the basis of cellulose. Other more preferredcarriers are polyacrylate or polystyrene based carriers having weaklybasic groups. Weakly basic anion exchangers are materials having primaryand/or secondary or/tertiary amino groups. They dissociate and haveexchange capability in acidic solutions. The materials having tertiaryamino groups have rather basic properties and they are also calledmedium basic anion exchangers. Preferably, phenolformaldehyde basedcarriers are used, such as commercial available Duolite™ A 568 (Rohm andHaas).

Preferably, the carrier is an anion exchange resin and the enzyme isimmobilized onto it by adsorption i.e. in a non-covalent manner. Thisenables the easy removal of the inactive enzyme and the subsequentreloading with fresh enzyme. Reloading of the carrier results incomplete recovery of the activity of the conjugate. This means that thecarrier material can be used for a considerable period of time before ithas to be replaced. Also, a treatment with a cross-linking agent, suchas glutaric dialdehyde can be performed to stabilise the immobilisedenzymes.

Furthermore, the current invention relates to a syrup containingisomalto-oligosaccharides with elongated chain length and obtainable bythe previously described process.

Additionally, the current invention relates to the use of the syrupcontaining isomalto-oligosaccharides with elongated chain length infood, beverages, feed, cosmetics or pharmaceutical products.

It can be used as a slowly or non-digestible oligosaccharides, a lowcalorie provider, a prebiotic, a mineral absorption promoting agent, anon-cariogenic agent, and/or a low glycemic index regulating syrup.

The specified syrup containing isomalto-oligosaccharides with elongatedchain length is defined as a carbohydrate material which provides,compared to maltose, a slower release and, therefore, a slowerabsorption into the body of glucose during transit through the smallintestine.

Applications of said specified syrup are many fold, i.e. they can beused to deliver sufficient carbohydrates to diabetic patients without asignificant raise of the serum glucose level. Also, the addition of thespecified syrup to the diet of elderly people confronted with a reducedglucose tolerance could have a beneficial effect.

In the domain of sports nutrition, said specified syrup could haveinteresting applications. Said syrup could supply an athlete with asteady and constant carbohydrate supply during physical exercise.Advantageously, the syrup may therefore be used as a component in sportsor energy beverages.

The present invention also provides the use of said syrup in food ordrink composition (=beverages). Typically, the food composition may be adiabetic food, a baby food, a diet food, for instance for sedentarypeople or a specially formulated food for people having a reducedglucose tolerance, for instance the elderly.

The presence of said syrup in food and drink compositions provides, asmentioned earlier, a slower release, and therefore a slower absorptioninto the body, of glucose. This is correlated with a low glycemic index.

The current invention has the following advantages:

-   -   isomalto-oligosaccharides with elongated chain length can be        prepared by a simple process,    -   it is not necessary to use a pure acceptor for the production of        isomalto-oligosaccharides with elongated chain length,    -   isomalto-oligosaccharides are directly reacted to obtain        isomalto-oligosaccharides with elongated chain length,    -   products obtainable by the current process show several health        beneficial effects which are useful in food, beverages, feed,        cosmetics and pharmaceutical products.

The current invention is illustrated by way of the following examples.

EXAMPLE 1

A syrup (IMO, Composition: 20.4% DP1, 26.0% DP2, 31.2% DP3, 11.7% DP4,3.9% DP5, 1.6% DP6, 0.8% DP7, 0.5% DP8, 0.3% DP9, 0.2% DP10, 3.4% DP>10;obtainable according to method of EP 0 875 585) was concentrated to 33%w/w and a sucrose solution at 33% w/w was added at different ratio's,going from 80/20 till 50/50 (v/v). The pH of the solutions was adjustedto 5.2 using 0.1 N HCl. All solutions were warmed up for 20 minutes at30° C. and 2 ml of a dextransucrase solution with an activity of 3.7U/ml was added to 20 ml of the prepared isomalto-oligosaccharide/sucrosesolutions.

The solution was incubated at 30° C. and samples were taken after 48 hrof incubation. The samples were diluted with 3 ml demineralised waterand put in a boiling water bath for 10 min. The solutions were filteredusing a 0.45 μm filter and analysed by HPLC. The results obtained aredisplayed in Table 1:

TABLE 1 Final Composition is expressed as % (HPLC-results) Ratiosubstrate IMO/sucrose IMO/sucrose IMO/sucrose IMO/sucrose Final comp.80/20 30/70 40/60 50/50 Glucose 17.9 16.6 15.6 15.2 Fructose 7.3 11.48.1 6.2 Sucrose 0.0 0.3 1.2 2.1 DP2 15.6 11.7 9.3 8.3 DP3 22.7 17.8 13.79.9 DP4 18.3 19.8 19.2 16.8 DP5 9.2 12.8 15.2 16.5 DP6 2.7 3.9 5.6 7.2DP7 1.2 1.3 1.8 2.7 DP8 0.5 0.5 0.6 1.1 DP9 0.3 0.3 0.3 0.4 DP10 0.3 0.10.1 0.3 DP > 10 4.0 4.0 3.9 4.4

While the substrate has a high concentration for DP2-DP3, after thereaction of 48 hours, a high concentration is observed in the DP4-DP5range (=isomalto-oligosaccharides with elongated chain length).

EXAMPLE 2

The syrup (IMO, Composition: 20.4% DP1, 26.0% DP2, 31.2% DP3, 11.7% DP4,3.9% DP5, 1.6% DP6, 0.8% DP7, 0.5% DP8, 0.3% DP9, 0.2% DP10, 3.4% DP>10obtainable according to method of EP 0 875 585) was concentrated to 33%w/w and a sucrose solution of 33% w/w was added at different ratio's,going from 80/20 till 50/50 (v/v). The pH of the solutions was adjustedto 5.2 using 0.1 N HCl. All solutions were warmed up for 20 minutes at30° C. and 2 ml of a alternansucrase solution (Leuconostoc mesenteroidesNRRL B-1355) with an activity of 6.6 U/ml was added.

The solution was incubated at 30° C. and samples were taken after 48 hrof incubation. The samples were diluted with 3 ml demineralised waterand put in a boiling water bath for 10 min. The solutions were filteredusing a 0.45 μm filter and analysed by HPLC. The results obtained aredisplayed in Table 2:

TABLE 2 Final Composition is expressed as % (HPLC-results) Ratiosubstrate IMO/sucrose IMO/sucrose IMO/sucrose IMO/sucrose Final comp.80/20 30/70 40/60 50/50 Glucose 19.7 17.4 15.3 8.8 Fructose 6.0 10.415.4 19.2 Sucrose 0.0 0.0 0.0 2.5 DP2 14.5 10.0 7.3 4.2 DP3 25.3 19.214.1 8.4 DP4 19.3 21.7 21.3 16.8 DP5 8.3 13.8 15.5 20.7 DP6 2.4 4.4 5.910.6 DP7 0.9 1.5 2.0 4.5 DP8 0.5 0.6 0.7 1.7 DP9 0.2 0.2 0.2 0.5 DP100.2 0.2 0.2 0.2 DP > 10 2.6 2.9 2.0 1.9

While the substrate has a high concentration for DP2-DP3, after thereaction of 48 hours, a high concentration is observed in the DP4-DP6range (=isomalto-oligosaccharides with elongated chain length).

In-Vitro Digestion of Syrup Prepared in Example 2

The IMO syrup (Composition: 20.4% DP1, 26.0% DP2, 31.2% DP3, 11.7% DP4,3.9% DP5, 1.6% DP6, 0.8% DP7, 0.5% DP8, 0.3% DP9, 0.2% DP10, 3.4% DP>10;obtainable according to method of EP 0 875 585) and the syrup obtainedin example 2 by the action of dextransucrase on a 50/50 IMO sucrose(v/v) solution, was incubated with porcine intestinal acetone powder at37° C. and pH6 for 2 hours.

Maltose was taken as reference. The results are displayed in Table 3:

TABLE 3 % dextrose released compared to Syrup reference Maltose 100 IMO26 Elongated IMO (example 2) 15.5

The results clearly demonstrate the slow digestibility of the syrupobtainable by the current invention.

1. A process of elongating the chain length ofisomalto-oligosaccharides, wherein said process comprises: (a)enzymatically converting a malto-oligosaccharide syrup with one or moreisomalto-oligosaccharide-producing enzymes into a carbohydrate syrupcontaining isomalto-oligosaccharides; (b) preparing a solutioncontaining the isomalto-oligosaccharides by adding sucrose to thecarbohydrate syrup from (a), wherein the ratio by volume (v/v) ofisomalto-oligosaccharides in the carbohydrate syrup from (a) to sucrosein said solution is in the range of from about 50:50 (v/v) to about30:70 (v/v); (c) adding at least one glucansucrase to said carbohydratesyrup or to said solution in (b); and (d) in the presence of saidglucansucrase, reacting the sucrose with the isomalto-oligosaccharidesin the solution, thereby producing a syrupy mixture wherein said syrupymixture contains isomalto-oligosaccharides having an elongated chainlength.
 2. The process of claim 1, wherein in step (a) theisomalto-oligosaccharide producing enzyme is transglucosidase.
 3. Theprocess of claim 1, wherein the process further comprises, between steps(a) and (b) removing glucose from the carbohydrate syrup bychromatographic purification.
 4. The process of claim 1, wherein theprocess further comprises chromatographically purifying the syrupymixture of step (d).
 5. The process of claim 1, wherein theenzymatically converting with one or moreisomalto-oligosaccharide-producing enzymes of step (a), the adding atleast one glucansucrase of step (c), and/or the reacting in the presenceof glucansucrase of step (d) is performed on a re-usable carrier.
 6. Theprocess of claim 1, wherein said at least one glucansucrase is selectedfrom the group consisting of dextransucrase, alternansucrase,mutansucrase, and mixtures thereof.
 7. The process of claim 1, whereinthe at least one glucansucrase is dextransucrase.
 8. The process ofclaim 1, wherein the at least one glucansucrase is alternansucrase. 9.The process of claim 1, wherein the at least one glucansucrase ismutansucrase.
 10. A process for elongating the chain length ofisomalto-oligosaccharides, the process comprising: (a) mixing sucroseand a carbohydrate syrup to form a solution, said carbohydrate syrupcontaining isomalto-oligosaccharides; (b) adding an enzyme to thesolution, to enzymatically catalyze a transfer reaction between saidsucrose and said isomalto-oligosaccharides, wherein the v/v ratio ofsaid sucrose to said isomalto-oligosaccharides is 5000 (v/v) or more andwherein the enzyme comprises at least one glucansucrase, therebyproducing a syrupy mixture containing isomalto-oligosaccharides havingan elongated chain length.
 11. The process of claim 10, wherein in saidprocess the sucrose content of the solution is 60% (v/v).
 12. Theprocess of claim 10, wherein in said process the sucrose content of thesolution is 70% (v/v).
 13. The process of claim 10, wherein said enzymeis in an immobilized enzyme.
 14. The process of claim 10, wherein instep (a) said mixing sucrose comprises mixing a 33% (w/w) solution ofsucrose.
 15. The process of claim 1, wherein in step (c) said at leastone glucansucrase is added to said carbohydrate syrup.
 16. The processof claim 1, wherein in step (b) the sucrose is a sucrose solution, andsaid sucrose solution is admixed with the carbohydrate syrup.
 17. Theprocess of claim 1, wherein in step (b), the adding of the sucrose tothe carbohydrate syrup comprises adding a solution of the sucrose to thecarbohydrate syrup, wherein the percent by weight (% (w/w)) sucrose inthe sucrose solution is equal to the % (w/w) isomalto-oligosaccharide inthe carbohydrate syrup.